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Abstract

CRISPR–Cas9 systems are bacterial adaptive immune systems that defend against infection by phages. Through the RNA-guided endonuclease activity of Cas9 they degrade double-stranded DNA with a protospacer adjacent motif (PAM) and sequences complementary to the guide RNA1,2,3,4,5. Recently, two anti-CRISPR proteins (AcrIIA2 and AcrIIA4 from Listeria monocytogenes prophages) were identified, both of which inhibit Streptococcus pyogenes Cas9 (SpyCas9) and L. monocytogenes Cas9 activity in bacteria and human cells6. However, the mechanism of AcrIIA2- or AcrIIA4-mediated Cas9 inhibition remains unknown. Here we report a crystal structure of SpyCas9 in complex with a single-guide RNA (sgRNA) and AcrIIA4. Our data show that AcrIIA2 and AcrIIA4 interact with SpyCas9 in a sgRNA-dependent manner. The structure reveals that AcrIIA4 inhibits SpyCas9 activity by structurally mimicking the PAM to occupy the PAM-interacting site in the PAM-interacting domain, thereby blocking recognition of double-stranded DNA substrates by SpyCas9. AcrIIA4 further inhibits the endonuclease activity of SpyCas9 by shielding its RuvC active site. Structural comparison reveals that formation of the AcrIIA4-binding site of SpyCas9 is induced by sgRNA binding. Our study reveals the mechanism of SpyCas9 inhibition by AcrIIA4, providing a structural basis for developing ‘off-switch’ tools for SpyCas9 to avoid unwanted genome edits within cells and tissues.

Acknowledgements

We thank J. He at Shanghai Synchrotron Radiation Facility (SSRF) for help with data collection. We thank J. Chai for critical reading of the manuscript. This research was funded by the National Natural Science Foundation of China grant no. 31422014, 31450001 and 31300605 to Z.H.

Author information

Author notes

De Dong

, Minghui Guo

& Sihan Wang

These authors contributed equally to this work.

Affiliations

HIT Center for Life Sciences, School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China

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Contributions

D.D., M.G. and S.W. expressed, purified, characterized and crystallized the AcrIIA4–SpyCas9–sgRNA complex with the help of S.W., Z.X., J.Y. and Z.X. D.D., M.G., Y.Z. and Z.H. carried out crystallographic studies. D.D., M.G. and Z.H. prepared the figures. D.D. and M.G. performed in vitro transcription of sgRNA, in vitro dsDNA cleavage, GST pull-down, gel filtration, microscale thermophoresis and electrophoretic mobility shift assay experiments with the help of S.W., Z.X., J.Y. and Z.X. Z.H., D.D., M.G. and S.W. wrote the paper. All authors contributed to the manuscript preparation. Z.H. designed the experiments.

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Editorial Summary

Anti-CRISPR protein blocks Cas9 activity

Bacteria possess a protective mechanism, the CRISPR system, to guard against invading phages. Phages have their own counteracting defence mechanism, as they encode anti-CRISPR proteins. Zhiwei Huang and colleagues have solved the structure of an anti-CRISPR protein, AcrIIA4, bound to a Cas9 and guide RNA complex. This structure reveals that the anti-CRISPR protein inhibits two processes. First, it inhibits Cas9 binding by mimicking a specific DNA sequence, known as PAM, which Cas9 uses to recognize its double-stranded DNA target. Second, it blocks the endonuclease active site of Cas9. This information suggests that it may be possible to develop 'off' switches to more precisely control Cas9-mediated genome engineering.